Liquid chromatography coupled on-line to flow cytometry for postcolumn homogeneous biochemical detection

The feasibility of flow cytometry as read-out principle for homogeneous cell- or bead-based assays coupled on-line to LC is demonstrated using digoxin-coated beads (Dig-Beads) and fluorescent-labeled anti-digoxin (AD-FITC) as model system. The assay is carried out in a postcolumn continuous-flow reaction detection system where the AD-FITC and Dig-Beads are simultaneously added to the eluate of an LC separation column. Binding of AD-FITC to Dig-Beads results in a constant amount of fluorescence associated with the beads, which is detected by the flow cytometer. The presence of active compounds, such as digoxin and its analogues, in the sample will results in a decrease of the AD-FITC-Dig-Bead complex and, consequently, in the bead-associated fluorescence. Hence, the bead-associated fluorescence detected is inversely related to the digoxin concentration. A data-handling algorithm was developed in-house for adequate analysis of raw data output from the flow cytometer. Various conditions that influence the performance of this novel LC-biochemical detection (LC-BCD) system were investigated to determine the optimal settings of the bead-based biochemical interaction. The optimized flow injection bead-based assay was capable of detecting very low concentrations of digoxigenin (0.5 nmol/L), digoxin (0.1 nmol/L), and gitoxigenin (50 nmol/L). The applicability of LC coupled on-line to flow cytometry was demonstrated by the individual detection of digoxin, digoxigenin, and gitoxigenin in a single LC analysis. The successful coupling of LC on-line to flow cytometry principally enables the use of a wide range of new homogeneous assay formats in LC-BCD, such as membrane-bound receptor assays, cell-binding assays, and functional cell-based assays. Next to the ability to use insoluble targets, and also multiplexing assays, i.e., performing a number of assays simultaneously, using color- or size-coded beads becomes at hand in LC-BCD.     

Improving the Sensitivity of Fluorescence‐Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads

In fluorescence‐based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay. Here, several widely used magnetic beads are photobleached and their autofluorescence is reduced to 1% of the initial value. Their autofluorescence properties, including their photobleaching decay rates and autofluorescence spectra pre‐ and post‐photobleaching, and the stability of the photobleaching over a period of two months are analyzed. The photobleached beads are stable over time and their surface functionality is retained. In a high‐sensitivity LX‐200 system using photobleached magnetic beads, human interleukin‐8 is detected with a threefold improvement in detection limit and signal‐to‐noise ratio over results achievable with nonbleached beads. Since many contemporary immunoassays rely on magnetic beads as capture surfaces, prebleaching the beads may significantly improve the analytical performance of these assays. Moreover, nonmagnetic beads with low autofluorescence are also successfully photobleached, suggesting that photobleaching can be applied to various capture surfaces used in fluorescence‐based assays.     

Simple bead assay for detection of live bacteria (Escherichia coli)

Bead assays are an important rapid microbial detection technology suitable for extremely low pathogen levels. We report a bead assay for rRNA extracted from Escherichia coli K12 that does not require amplification steps and has readout on an Agilent 2100 Bioanalyzer flow cytometry system. Our assay was able to detect 125 ng of RNA, which is 16 times less than reported earlier. The specificity was extremely high, with no binding to a negative control organism (Bacillus subtilis). We discuss challenges faced during optimization of the key assay components, such as varying amounts of RNA in the samples, number of beads, aggregation, and reproducibility.     

Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ～1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.     

Tandem dye acceptor used to enhance upconversion fluorescence resonance energy transfer in homogeneous assays

In fluorescence resonance energy transfer (FRET)-based assays, spectral separation of acceptor emission from donor emission is a common problem affecting the assay sensitivity. The challenge derives from small Stokes shifts characteristic to conventional fluorescent dyes resulting in leakage of donor emission to the measurement window intended only to collect the acceptor emission. We have studied a FRET-based homogeneous bioaffinity assay utilizing a tandem dye acceptor with a large pseudo-Stokes shift (139 nm). The tandem dye was constructed using B-phycoerythrin as an absorber and multiple Alexa Fluor 680 dyes as emitters. As a donor, we employed upconverting phosphor particles, which uniquely emit at visible wavelengths under low-energy infrared excitation enabling the fluorescence measurements free from autofluorescence even without time-resolved detection. With the tandem dye, it was possible to achieve four times higher signal from a single binding event compared to the conventional Alexa Fluor 680 dye alone. Tandem dyes are widely used in cytometry and other multiplex purposes, but their applications can be expanded to fluorescence-based homogeneous assays. Both the optimal excitation and emission wavelengths of tandem dye can be tuned to a desired region by choosing appropriate fluorophores enabling specifically designed acceptor dyes with large Stokes shift.     

Automated bead alignment apparatus using a single bead capturing technique for fabrication of a miniaturized bead-based DNA probe array

We have developed an automated bead alignment apparatus for fabricating a bead-based DNA probe array inside a capillary. The apparatus uses 16 micro vacuum tweezers to extract single beads from among a large amount of beads in bead stock wells. It then manipulates single beads into the probe array capillaries. Single 100-microm-diameter beads were successfully extracted from the water-contained bead-stock well by the vacuum tweezers, which have inner and outer diameters of 50 and 150 microm. An interesting aspect is that unexpected extra beads adsorbed on the outer wall of the vacuum tweezers can be removed using the surface tension force between the water and the atmosphere. In testing the total performance of this apparatus, the DNA probe arrays with 10 sets of probe-conjugated beads and 2 plain beads were produced in the intended order in the capillaries. The time needed to align the 12 beads was 10 min, and the 16 bead arrays were fabricated simultaneously. After hybridization experiments using these fabricated DNA probe arrays, fluorescence from each bead was clearly observed.     

Multiplexed flow cytometric immunoassay for influenza virus detection and differentiation

Microsphere-based immunoassay by flow cytometry has gained popularity lately in protein detection and infectious disease diagnosis due to its capacity for multiplexed analysis and simple assay format. Here, we demonstrated the power of microsphere-based immunoassay for high-sensitivity detection and accurate differentiation of influenza viruses. The effects of sample volume and bead number on the assay sensitivity of viral antigen detection were studied. Compared to enzyme-linked immunosorbent assays, flow-based bead assays provided approximately 10-fold lower detection limit for viral particle detection and performed similarly for recombinant viral hemagglutinin protein detection. A four-plexed assay for influenza virus typing and influenza B virus sublineage characterization was developed to demonstrate the potential for multiplexed viral antigen detection and differentiation.     

Efficient detection of single DNA fragments in flowing sample streams by two-photon fluorescence excitation

This paper reports the demonstration of efficient single molecule detection in flow cytometry by two-photon fluorescence excitation. We have used two-photon excitation (TPE) to detect single DNA fragments as small as 383 base pairs (bp) labeled with the intercalating dye, POPO-1, at a dye:nucleotide ratio of 1:5. TPE of the dye-DNA complexes was accomplished using a mode-locked, 120 fs pulse width Ti:sapphire laser operating at 810 nm. POPO-1 labeled DNA fragments of 1.1 kilobase pairs (kbp) and larger were sequentially detected in our flow cytometry system with a detection efficiency of nearly 100%. The detection efficiency for the 383 bp DNA fragments was approximately 75%. We also demonstrate the ability to distinguish between different sized DNA fragments in a mixture by their individual fluorescence burst sizes by TPE. These studies indicate that using TPE for single molecule flow cytometry experiments lowers the intensity of the background radiation by approximately an order of magnitude compared to one-photon excitation, due to the large separation between the excitation and emission wavelengths in TPE.     

Ultrasensitive detection of cancer biomarkers in the clinic by use of a nanostructured microfluidic array

Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5-50 fg·mL(-1) range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400,000 enzyme labels and ~100,000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linked immunosorbent assays (ELISA) for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the four protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity of 89% and specificity of 98% for oral cancer detection, demonstrating high diagnostic utility. The low-cost, easily fabricated immunoarray provides a rapid serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers.     

Direct manipulation and observation of the rotational motion of single optically trapped microparticles and biological cells in microvortices

This paper describes a method for manipulating and monitoring the rotational motion of single, optically trapped microparticles and living cells in a microvortex. To induce rotation, we placed the microparticle at the center of rotation of the vortex and used the recirculating fluid flow to drive rotation. We have monitored the rotation of single beads (which ranged in diameter from a few micrometers to tens of micrometers) and living cells in a microvortex. To follow the rotation of a smooth and symmetrically shaped bead, we first ablated a small region ( approximately 1 microm) on the bead. An Ar(+) laser was then tightly focused ( approximately 0.5-microm spot size) onto the bead, and rotation was tracked by recording changes in the level of backscattered laser light as the ablated region repeatedly transited the laser focus. Using this method, we have followed bead rotation that varied in frequency from 0.15 to 100 Hz and have studied the effect of bead diameter on the rate of rotation at a given fluid flow rate. To monitor the rotation of single living cells, we selectively stained portions of B-lymphocytes with the fluorescent dye DiOC(6). We observed rotation by following changes in the fluorescence signal as the dye-stained region transited the laser focal volume. This technique provides a simple and sensitive method for controlling and monitoring the rotational motion of microparticles in a microfluidic environment.     

Spectroscopic characterization of coumarin-stained beads: quantification of the number of fluorophores per particle with solid-state 19F-NMR and measurement of absolute fluorescence quantum yields

The rational design of nano- and micrometer-sized particles with tailor-made optical properties for biological, diagnostic, and photonic applications requires tools to characterize the signal-relevant properties of these typically scattering bead suspensions. This includes methods for the preferably nondestructive quantification of the number of fluorophores per particle and the measurement of absolute fluorescence quantum yields and absorption coefficients of suspensions of fluorescent beads for material performance optimization and comparison. Here, as a first proof-of-concept, we present the first time determination of the number of dye molecules per bead using nondestructive quantitative ((19)F) NMR spectroscopy and 1000 nm-sized carboxylated polystyrene particles loaded with varying concentrations of the laser dye coumarin 153 containing a CF(3) group. Additionally, the signal-relevant optical properties of these dye-loaded particles were determined in aqueous suspension in comparison to the free dye in solvents of different polarity with a custom-built integrating sphere setup that enables spectrally resolved measurements of emission, transmission, and reflectance as well absolute fluorescence quantum yields. These measurements present an important step toward absolute brightness values and quantitative fluorescence analysis with particle systems that can be exploited, for example, for optical imaging techniques and different fluorescence assays as well as for the metrological traceability of fluorescence methods.     

Electrochemiluminescence enzyme immunoassays for TNT and pentaerythritol tetranitrate

Electrochemiluminescence enzyme immunoassays for 2,4,6-trinitrotoluene (TNT) and pentaerythritol tetranitrate (PETN) are described. The latter is, to the best of our knowledge, the first report of an immunoassay for PETN. Haptens corresponding to these explosives were covalently attached to high-affinity dextran-coated paramagnetic beads. The beads were mixed with the corresponding Fab fragments and the sample. After adding a second HRP-labeled antispecies-specific antibody, the mixture was pumped into an electrochemiluminometer where beads were concentrated on the working electrode magnetically. The amount of analyte in the sample was determined by measuring light emission when H2O2 was generated electrochemically in the presence of luminol and an enhancer. The detection limits for TNT and PETN were 0.11 and 19.8 ppb, respectively. Details of bead preparation and performance are given. The increase in sensitivity obtained when Fab fragments are used instead of whole antibodies is explained, and the implications of this observation for nanoparticle-based assays are discussed.     

Highly fluorescent dye-doped silica nanoparticles increase flow cytometry sensitivity for cancer cell monitoring

Early and accurate diagnosis and treatment of cancer depend on rapid, sensitive, and selective detection of tumor cells. Current diagnosis of cancers, especially leukemia, relies on histology and flow cytometry using single dye-labeled antibodies. However, this combination may not lead to high signal output, which can hinder detection, especially when the probes have relatively weak affinities or when the receptor is expressed in a low concentration on the target cell surface. To solve these problems, we have developed a novel method for sensitive and rapid detection of cancer cells using dye-doped silica nanoparticles (NPs) which increases detection sensitivity in flow cytometry analyses between 10- and 100-fold compared to standard methods. Our NPs are ∼60 nm in size and can encapsulate thousands of individual dye molecules within their matrix. We have extensively investigated surface modification strategies in order to make the NPs suitable for selective detection of cancer cells using flow cytometry. The NPs are functionalized with polyethylene glycol (PEG) to prevent nonspecific interactions and with neutravidin to allow universal binding with biotinylated molecules. By virtue of their reliable and selective detection of target cancer cells, these NPs have demonstrated their promising usefulness in conventional flow cytometry. Moreover, they have shown low background signal, high signal enhancement, and efficient functionalization, either with antibody- or aptamer-targeting moieties. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Determination of carcinoembryonic antigen in human sera by integrated bead-bed immunoassay in a microchip for cancer diagnosis

A bead-bed immunoassay system was structured on a microchip and applied to determine carcinoembryonic antigen (CEA), which is a commonly used marker of colon cancer. Polystyrene beads precoated with anti-CEA antibody were introduced into a microchannel, and then a serum sample containing CEA, the first antibody, and the second antibody conjugated with colloidal gold were reacted successively. The resulting antigen-antibodies complex, fixed on the bead surface, was detected using a thermal lens microscope (TLM). A highly selective and sensitive determination of an ultratrace amount of CEA in human sera was made possible by a sandwich immunoassay system that needs three antibodies for an assay. A detection limit dozens of times lower than the conventional ELISA was achieved. Moreover, when serum samples for 13 patients were assayed with this system, there was a high correlation (r = 0.917) with the conventional ELISA. The integration reduced the time necessary for the antigen-antibody reaction to approximately 1%, thus shortening the overall analysis time from 45 h to 35 min. Moreover, troublesome operations required for conventional heterogeneous immunoassays could be much simplified. This microchip-based diagnosis system is the first microchip-based system that is practically useful for clinical diagnoses with short analysis time, high sensitivity, and easy procedures.     

A microchip-based multianalyte assay system for the assessment of cardiac risk

The development of a novel chip-based multianalyte detection system with a cardiac theme is reported. This work follows the initial reports of "electronic taste chips" whereby multiple solution-phase analytes such as acids, bases, metal cations, and biological cofactors were detected and quantitated. The newly fashioned "cardiac chip" exploits a geometry that allows for isolation and entrapment of single polymeric spheres in micromachined pits while providing to each bead the rapid introduction of a series of reagents/washes through microfluidic structures. The combination of these miniaturized components fosters the completion of complex assays with short analysis times using small sample volumes. Optical signals derived from single beads are used to complete immunological tests that yield outstanding assay characteristics. The power and utility of this new methodology is demonstrated here for the simultaneous detection of the cardiac risk factors, C-reactive protein and interleukin-6, in human serum samples. This demonstration represents the first important step toward the development of a useful cardiac chip that targets numerous risk factors concurrently and one that can be customized readily for specific clinical settings.     

Application of ganglioside-sensitized liposomes in a flow injection immunoanalytical system for the determination of cholera toxin

Cholera, an acute infectious disease associated with water and seafood contamination, is caused by the bacterium Vibrio cholerae, which lives and colonizes in the small intestine and secretes cholera toxin (CT), a causative agent for diarrhea in humans. Based on earlier lateral flow assays, a flow injection liposome immunoanalysis (FILIA) system with excellent sensitivity was developed in this study for the determination of CT at zeptomole levels. Ganglioside (GM1), found to have specific affinity toward CT, was inserted into the phospholipid bilayer during the liposome synthesis. These GM1-sensitized, sulforhodamine B (SRB) dye-entrapping liposomes were used as probes in the FILIA system. Anti-CT antibodies were immobilized in its microcapillary. CT was detected by the formation of a sandwich complex between the immobilized antibody and GM1 liposomes. During the assay, the sample was introduced first into the column, and then liposomes were injected to bind to all CT captured by the antibody in the microcapillary. Subsequently, the SRB dye molecules were released from the bound liposomes via the addition of the detergent octyl glucopyranoside. The released dye molecules were transported to a flow-through fluorescence detector for quantification. The FILIA system was optimized with respect to flow rate, antibody concentration, liposome concentration, and injected sample volume. The calibration curve for CT had a linear range of 10-16 to 10-14 g mL-1. The detection limit of this immunosensor was 6.6 x 10(-17) g mL-1 in 200-microL samples (equivalent to 13 ag or 1.1 zmol).     

Magnetic bead based immunoassay for autonomous detection of toxins

We are developing an automated system for the simultaneous, rapid detection of a group of select agents and toxins in the environment. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads and a photoactive porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactive group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags. Released eTags are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated in a flow-through format with higher LODs of 32 ng/mL (or 640 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.     

High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets

A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (microDG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the microDG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation. After bulk PCR amplification, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry. DNA targets ranging in size from 380 to 1139 bp at single molecule concentrations are quantitatively amplified using SCGA. Long-range sequencing results from beads each carrying approximately 100 amol of a 624 bp product demonstrate that these amplicons are competent for achieving attomole-scale Sanger sequencing from a single bead and for advancing pyrosequencing read-lengths. Successful single cell analysis of the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene in human lymphocyte cells and of the gyr B gene in bacterial Escherichia coli K12 cells establishes that SCGA will also be valuable for performing high-throughput genetic analysis on single cells.